PROJECT SUMMARY Preeclampsia (PE) is a devastating hypertensive disorder of pregnancy that is a leading cause of maternal and fetal mortality and morbidity worldwide. It affects 2-10% of women and accounts for 16% of maternal deaths related to childbirth in developed countries. PE is difficult to study because it is a multifactorial disorder with varying molecular mechanisms associated with the development of the spectrum of clinical symptoms associated with early onset, late onset, and severe PE. A personalized medicine approach applied to dissecting the underlying mechanisms of PE may be beneficial to reduce clinical incidence of this devastating syndrome. The placenta plays a key role in the development of PE, leading to widespread maternal endothelial dysfunction, hypertension, and systemic multi-organ failure in PE. Extracellular vesicles (EVs) containing protein, RNA, and lipid cargo are continuously extruded from the placenta, and are capable of interacting with maternal organs including the kidney. PE is primarily associated with placental and renal dysfunction, and PE is the most common cause of acute kidney injury during pregnancy. However, no studies have investigated the potential of placenta-derived RNA cargo as a link between placental and renal dysfunction in PE. Urinary EVs are derived from multiple tissue types and represent a trove of biomarkers that are increasingly being utilized to diagnose renal disorders. Further, urine samples can be obtained throughout pregnancy non-invasively and could potentially be utilized to identify biomarkers related to placental dysfunction in PE. In the proposed research, during the mentored phase, cutting-edge RNA-Seq technology coupled with computational biological and machine learning approaches will be applied to profile the transcriptome of urinary EVs in women with PE compared to normal pregnancy. Preliminary data indicates that it is possible to isolate and profile the transcriptome of urinary EVs from maternal urine throughout normal gestation, and that placenta-derived and placenta-specific mRNA and miRNA can be detected within the urinary EV population. This presents a novel technique that has potential to identify biomarkers as well as provide information on placental dysfunction in PE in a non-invasive manner. During the independent phase, the candidate will utilize an in vitro approach to investigate the effect of uptake of placenta-derived EVs with miRNA cargo associated with PE on the function of proximal tubule epithelial cells and cortical collecting duct cells. These two renal-specific cell types are involved in tubular reabsorption in the nephron, a process that is compromised leading to increased excretion of protein in the urine in some preeclamptic pregnancies. This proposal is multidisciplinary, utilizing basic biology, clinical research, and high-performance computing applied to investigating placental dysfunction in PE. These experiments are significant because they will generate novel information on the role of placenta- derived EVs in renal dysfunction in PE, as well as point the way towards preventative and therapeutic targets that may be transformative and clinically relevant.